Discovery of novel therapeutics for fungal pathogens Abstract Fungal central nervous system (CNS) infection continues be an important cause of mortality and morbidity. Cryptococcus neoformans (C. neoformans) and Candida albicans (C. albicans) represent the important fungal pathogens causing CNS infection. Successful treatment of C. neoformans and C. albicans CNS infection is compromised by development of antimicrobial resistance and limited repertoire of antifungal drugs against resistant C. neoformans and C. albicans infection. This application, submitted in response to RFA-AI-15-054, proposes to develop early stage translational research focused on the discovery and development of novel therapeutics against CNS infection caused by eukaryotic fungal pathogens, C. neoformans and C. albicans. Recent studies have shown that CNS-infecting pathogens exhibit the ability to penetrate the blood-brain barrier, the essential step in the development of CNS infection. Since C. neoformans and C. albicans dissemination to the brain is likely to involve their penetration of the blood-brain barrier, we developed the in vitro blood-brain barrier model with human brain microvascular endothelial cells (HBMEC) to investigate C. neoformans and C. albicans penetration of the blood-brain barrier. The HBMEC monolayer, upon cultivation on collagen-coated Transwell inserts, exhibits morphological and functional properties of tight junction formation and a polarized monolayer, a unique property of the blood-brain barrier endothelial cells. We showed that C. neoformans and C. albicans strains exhibited the ability to penetrate the blood-brain barrier. We hypothesize that targeting C. neoformans and C. albicans penetration of the blood-brain barrier provides a novel approach for discovery of novel therapeutics for fungal CNS infection. This hypothesis is supported by our preliminary identification of montelukast (an antagonist of cysteinyl leukotriene type 1 receptor or CysLT1) and gefitinib (an inhibitor of epidermal growth factor receptor or EGFR) that inhibited C. neoformans and C. albicans penetration of the blood-brain barrier. CysLT1 and EGFR have not been previously recognized for their contribution to fungal CNS infection and represent novel therapeutic targets for C. neoformans and C. albicans CNS infection. Determination and validation of CysLT1 and EGFR for their contribution to C. neoformans and C. albicans penetration of the blood-brain barrier will, therefore, facilitates discovery of novel host-specific therapeutic targets common to C. neoformans and C. albicans, which is consistent with the FOA of RFA-AI-15-054.